Episodes of severe low blood sugar (hypoglycemia) are a major problem for Type 1 diabetic patients. Normally, the brain senses hypoglycemia and triggers a "fight-or-flight" stress response to rapidly increase blood sugar and prevent a seizure or coma. Unfortunately, people with Type 1 diabetes do not detect low blood glucose as well as non-diabetic individuals and their ability to defend against it is severely impaired. Therefore, they are at a greater risk of experiencing severe hypoglycemic episodes. Hypoglycemia is the limiting step in the treatment of diabetes and precludes against the benefits linked to intensive glucose control. Due to these consequences, a major research initiative of the NIDDK is to prevent or reduce hypoglycemia in Type 1 diabetics, specifically by defining the "mechanisms and modulators" of brain glucose sensing. This proposal is designed to study the role and mechanism by which insulin regulates brain glucose sensing. Key evidence suggests that insulin acts in the brain to regulate glucose homeostasis, CNS glucose sensing, and the counterregulatory response (CRR) to hypoglycemia, but the site and method of CNS insulin action are still unknown. This study will 1) investigate whether insulin acts on hypothalamic neurons to regulate brain glucose sensing and 2) determine how insulin regulates glucose sensing by evaluating its effects on key glucose sensors and CNS glucose uptake. First, taking advantage of a genetic mouse model that lacks CNS insulin action (NIRKO mouse), this study will assess whether CNS insulin action regulates the brain's ability to detect and respond to hypoglycemia. The CRR and neuronal activation in response to hypoglycemia will be measured via hypoglycemic clamp studies and c-fos immunostaining. Further, to clarify a mechanism of CNS insulin action, this study will also assess whether insulin regulates key glucose sensors and/or CNS glucose uptake. Glucose sensing proteins, including glucose transporters (GLUTs) and glucokinase (GK), will be assayed for altered expression in the NIRKO mouse via standard immunohistochemical techniques. Similarly, to detect changes in brain glucose uptake, regional brain glucose utilization will be evaluated using a radioisotope assay. Overall, this study will provide new insights into insulin's role in regulating CNS glucose sensing and the hypoglycemic counterregulatory response. Because intensive insulin therapy increases the risk of severe hypoglycemia (low blood glucose episode), hypoglycemia impedes tight glucose management in diabetic patients. Therefore, understanding how the brain regulates the stress response to hypoglycemia is critical to devise therapies to combat hypoglycemia.